This study provides important new insights on how to achieve high sulfur selectivities and stable gas biodesulfurization process operation in the presence of both methanethiol and H₂S in the feed gas. On the basis of previous research, we hypothesized that a dual bioreactor lineup (with an added anaerobic bioreactor) would favor sulfur-oxidizing bacteria (SOB) that yield a higher sulfur selectivity. Therefore, the focus of the present study was to enrich thiol-resistant SOB that can withstand methanethiol, the most prevalent and toxic thiol in sulfur-containing industrial off gases. In addition, the effect of process conditions on the SOB population dynamics was investigated. The results confirmed that thiol-resistant SOB became dominant with a concomitant increase of the sulfur selectivity from 75 mol% to 90 mol% at a loading rate of 2 mM S methanethiol day⁻¹. The abundant SOB in the inoculum – Thioalkalivibrio sulfidiphilus – was first outcompeted by Alkalilimnicola ehrlichii after which Thioalkalibacter halophilus eventually became the most abundant species. Furthermore, we found that the actual electron donor in our lab-scale biodesulfurization system was polysulfide, and not the primarily supplied sulfide.